The regulation of p53 dynamics in response to DNA damage

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The regulation of p53 dynamics in response to DNA
damage

• Eric Batchelor
• Lahav Lab
• Department of Systems Biology
• Harvard Medical School

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How does the structure of the signaling
network shape the dynamic response?
What is the role of temporal dynamics of
signaling events in cellular decisions?
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The p53 Network Bow-tie Signaling Architecture
p53
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Previous studies reported damped p53
oscillations in response to IR
Lev Bar-Or et al, PNAS, 2000
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Fluorescent protein fusions to monitor the
network dynamics in individual cells
pMT-I
p53
CFP
hygroR
pMdm2
Mdm2
YFP
NeoR
3.5 kb
P53-CFP
Mdm2-YFP
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p53 dynamics after DNA damage (DSBs)

• Cells show undamped p53 pulses

• The mean amplitude and duration of the pulses do
  not depend of the amount of DNA damage

• The number cells in the population that pulse
  increases with DNA damage

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Cancer Research, 2006
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What is the mechanism regulating undamped p53
pulses in response to IR?
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p53 oscillations are intrinsic to the p53/Mdm2
loop
p53 oscillations are triggered by pulses of the
signal
DSBs
Double Strand Breaks
ATM-P
Chk2-P
p53
Mdm2
Geva-Zatorsky et al., Mol Sys Bio 2006
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Phosphorylated ATM shows pulses in Western blots
following DNA damage
Double Strand Breaks
Time (hrs)
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5
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9
8
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ATM-P(S1981)
ATM-P
Tubulin
Chk2-P
p53
ATM-P
Mdm2
Time (hr)
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Phosphorylated Chk2 shows pulses in Western
blots following DNA damage
Double Strand Breaks
Time (hrs)
0
1
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5
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7
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10
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Chk2-P(T68)
ATM-P
Chk2
Tubulin
Chk2-P
p53
Chk2-P
Mdm2
Time (hr)
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The mechanism regulating p53 pulses
What represses ATM-P and Chk2-P levels between
pulses?
Double strand Breaks
ATM-P
Chk2-P
?
p53
Mdm2
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Chk2-P pulses depend on p53
Chk2-P
Time (hrs)
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Chk2-P
Chk2
tubulin
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p53 over-expression inhibits Chk2 activation
Chk2-P
p53
Nutlin3
Vassilev et al. Science 2004
Mdm2
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The mechanism of p53 pulses
Are repeated ATM-P/Chk2-P pulses required for
series of p53 pulses?
Double strand Breaks
ATM-P
Chk2-P
p53
?
Mdm2
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Are repeated ATM-P/Chk2-P pulses required for
the series of p53 pulses?
Wortmannin
Chk2-P
Time (hrs)
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5
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Chk2-P
Chk2
p53
Wortmannin
Time (hrs)
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p53
Initial transient activation of p53 by ATM and
Chk2 is insufficient to drive multiple,
autonomous p53 pulses by the p53-Mdm2 module
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Model of recurrent initiation
Signal
Inhibitor
p53
Mdm2
Expansion of Geva-Zatorsky, Nitzan Rosenfeld et
al. Mol Sys Bio 2006
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Wip1 identified as the putative inhibtor

• Inhibitor should initially be at relatively low
  levels
• Inhibitor levels will undergo pulses, with pulse
  valleys at levels elevated with respect to
  basal level.

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Time (h)
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6.5
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p53
0.8
Wip1
0.6
Wip1
0.4
0.2
tubulin
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Time (h)
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Predicted Wip1 RNAi Effect
Depletion of Wip1 predicted to increase p53
pulse duration
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Effect of Wip1 RNAi on p53 in Single Cells
control RNAi
Wip1 RNAi
Wip1 important for maintaining constant pulse
amplitude and duration as a function of
g-irradiation dose
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Varied p53 Dynamic Responses to DNA Damage
p53
Mdm2
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p53 Shows Graded Pulses in Response to UV
Radiation
2 J/m2 UV
8 J/m2 UV
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p53-CFP Fluorescence
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Time (hrs)
Time (hrs)
p53 shows pulses that change in amplitude and
duration as a function of UV-irradiation dose
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Future directions

• How does Wip1 contribute to constant p53 pulse
  amplitude independently of irradiation dose?
• What is the p53 dynamic response to other DNA
  damaging stimuli? Non-DNA damaging stimuli?
• Can we synthetically perturb the system to
  switch between dynamic responses?
• Can we use this system as a model for
  bioengineered oscillators / pulse generators?
• How do the responses relate to p53 activity?

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Acknowledgements
Galit Lahav and the other Lahavians Irun
Bhan Madhulika Jain Ran Kafri Alex Löwer
Caroline Mock Dave Nelson Sara
Thiebaud Jared Toettcher Ketki Verkhedkar
Jeremy Gunawardena Roy Kishony